Nuclear magnetic resonance spectroscopy, most commonly known as NMR spectroscopy or magnetic resonance spectroscopy (MRS), is a spectroscopic technique to observe local magnetic fields around atomic nuclei. The sample is placed in a magnetic field and the NMR signal is produced by excitation of the nuclei sample with radio waves into nuclear magnetic resonance, which is detected with sensitive radio receivers. The intramolecular magnetic field around an atom in a molecule changes the resonance frequency, thus giving access to details of the electronic structure of a molecule and its individual functional groups. As the fields are unique or highly characteristic to individual compounds, in modern organic chemistry practice, NMR spectroscopy is the definitive method to identify monomolecular organic compounds. Similarly, biochemists use NMR to identify proteins and other complex molecules. Besides identification, NMR spectroscopy provides detailed information about the structure, dynamics, reaction state, and chemical environment of molecules. The most common types of NMR are proton and carbon-13 NMR spectroscopy, but it is applicable to any kind of sample that contains nuclei possessing spin.
Solid-state NMR (ssNMR) spectroscopy is a special type of nuclear magnetic resonance (NMR) spectroscopy, characterized by the presence of anisotropic interactions. Compared to the more common solution NMR spectroscopy, ssNMR usually requires additional hardware for high-power radio-frequency irradiation and magic-angle spinning.
An intrinsically disordered protein (IDP) is a protein that lacks a fixed or ordered three-dimensional structure. IDPs range from fully unstructured to partially structured and include random coils, (pre-)molten globules, and large multi-domain proteins connected by flexible linkers. They are one of the main types of protein along with globular, fibrous and membrane proteins.
Nuclear magnetic resonance spectroscopy of proteins is a field of structural biology in which NMR spectroscopy is used to obtain information about the structure and dynamics of proteins, and also nucleic acids, and their complexes. The field was pioneered by Richard R. Ernst and Kurt Wüthrich at the ETH, and by Ad Bax, Marius Clore, and Angela Gronenborn at the NIH, among others. Structure determination by NMR spectroscopy usually consists of several phases, each using a separate set of highly specialized techniques. The sample is prepared, measurements are made, interpretive approaches are applied, and a structure is calculated and validated.
The heteronuclear single quantum coherence or heteronuclear single quantum correlation experiment, normally abbreviated as HSQC, is used frequently in NMR spectroscopy of organic molecules and is of particular significance in the field of protein NMR. The experiment was first described by Geoffrey Bodenhausen and D. J. Ruben in 1980. The resulting spectrum is two-dimensional (2D) with one axis for proton (1H) and the other for a heteronucleus, which is usually 13C or 15N. The spectrum contains a peak for each unique proton attached to the heteronucleus being considered. The 2D HSQC can also be combined with other experiments in higher-dimensional NMR experiments, such as NOESY-HSQC or TOCSY-HSQC.
The residual dipolar coupling between two spins in a molecule occurs if the molecules in solution exhibit a partial alignment leading to an incomplete averaging of spatially anisotropic dipolar couplings.
Carbohydrate NMR Spectroscopy is the application of nuclear magnetic resonance (NMR) spectroscopy to structural and conformational analysis of carbohydrates. This method allows the scientists to elucidate structure of monosaccharides, oligosaccharides, polysaccharides, glycoconjugates and other carbohydrate derivatives from synthetic and natural sources. Among structural properties that could be determined by NMR are primary structure, saccharide conformation, stoichiometry of substituents, and ratio of individual saccharides in a mixture. Modern high field NMR instruments used for carbohydrate samples, typically 500 MHz or higher, are able to run a suite of 1D, 2D, and 3D experiments to determine a structure of carbohydrate compounds.
The Re-referenced Protein Chemical shift Database (RefDB) is an NMR spectroscopy database of carefully corrected or re-referenced chemical shifts, derived from the BioMagResBank (BMRB). The database was assembled by using a structure-based chemical shift calculation program to calculate expected protein (1)H, (13)C and (15)N chemical shifts from X-ray or NMR coordinate data of previously assigned proteins reported in the BMRB. The comparison is automatically performed by a program called SHIFTCOR. The RefDB database currently provides reference-corrected chemical shift data on more than 2000 assigned peptides and proteins. Data from the database indicates that nearly 25% of BMRB entries with (13)C protein assignments and 27% of BMRB entries with (15)N protein assignments require significant chemical shift reference readjustments. Additionally, nearly 40% of protein entries deposited in the BioMagResBank appear to have at least one assignment error. Users may download, search or browse the database through a number of methods available through the RefDB website. RefDB provides a standard chemical shift resource for biomolecular NMR spectroscopists, wishing to derive or compute chemical shift trends in peptides and proteins.
Triple resonance experiments are a set of multi-dimensional nuclear magnetic resonance spectroscopy (NMR) experiments that link three types of atomic nuclei, most typically consisting of 1H, 15N and 13C. These experiments are often used to assign specific resonance signals to specific atoms in an isotopically-enriched protein. The technique was first described in papers by Ad Bax, Mitsuhiko Ikura and Lewis Kay in 1990, and further experiments were then added to the suite of experiments. Many of these experiments have since become the standard set of experiments used for sequential assignment of NMR resonances in the determination of protein structure by NMR. They are now an integral part of solution NMR study of proteins, and they may also be used in solid-state NMR.
GeNMR method is the first fully automated template-based method of protein structure determination that utilizes both NMR chemical shifts and NOE -based distance restraints.
Protein Structure Evaluation Suite & Server (PROSESS) is a freely available web server for protein structure validation. It has been designed at the University of Alberta to assist with the process of evaluating and validating protein structures solved by NMR spectroscopy.
CS23D is a web server to generate 3D structural models from NMR chemical shifts. CS23D combines maximal fragment assembly with chemical shift threading, de novo structure generation, chemical shift-based torsion angle prediction, and chemical shift refinement. CS23D makes use of RefDB and ShiftX.
Conformational ensembles, also known as structural ensembles are experimentally constrained computational models describing the structure of intrinsically unstructured proteins. Such proteins are flexible in nature, lacking a stable tertiary structure, and therefore cannot be described with a single structural representation. The techniques of ensemble calculation are relatively new on the field of structural biology, and are still facing certain limitations that need to be addressed before it will become comparable to classical structural description methods such as biological macromolecular crystallography.
The chemical shift index or CSI is a widely employed technique in protein nuclear magnetic resonance spectroscopy that can be used to display and identify the location as well as the type of protein secondary structure found in proteins using only backbone chemical shift data The technique was invented by Dr. David Wishart in 1992 for analyzing 1Hα chemical shifts and then later extended by him in 1994 to incorporate 13C backbone shifts. The original CSI method makes use of the fact that 1Hα chemical shifts of amino acid residues in helices tends to be shifted upfield relative to their random coil values and downfield in beta strands. Similar kinds of upfield/downfiled trends are also detectable in backbone 13C chemical shifts.
Protein chemical shift prediction is a branch of biomolecular nuclear magnetic resonance spectroscopy that aims to accurately calculate protein chemical shifts from protein coordinates. Protein chemical shift prediction was first attempted in the late 1960s using semi-empirical methods applied to protein structures solved by X-ray crystallography. Since that time protein chemical shift prediction has evolved to employ much more sophisticated approaches including quantum mechanics, machine learning and empirically derived chemical shift hypersurfaces. The most recently developed methods exhibit remarkable precision and accuracy.
Nuclear magnetic resonance chemical shift re-referencing is a chemical analysis method for chemical shift referencing in biomolecular nuclear magnetic resonance (NMR). It has been estimated that up to 20% of 13C and up to 35% of 15N shift assignments are improperly referenced. Given that the structural and dynamic information contained within chemical shifts is often quite subtle, it is critical that protein chemical shifts be properly referenced so that these subtle differences can be detected. Fundamentally, the problem with chemical shift referencing comes from the fact that chemical shifts are relative frequency measurements rather than absolute frequency measurements. Because of the historic problems with chemical shift referencing, chemical shifts are perhaps the most precisely measurable but the least accurately measured parameters in all of NMR spectroscopy.
Protein chemical shift re-referencing is a post-assignment process of adjusting the assigned NMR chemical shifts to match IUPAC and BMRB recommended standards in protein chemical shift referencing. In NMR chemical shifts are normally referenced to an internal standard that is dissolved in the NMR sample. These internal standards include tetramethylsilane (TMS), 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) and trimethylsilyl propionate (TSP). For protein NMR spectroscopy the recommended standard is DSS, which is insensitive to pH variations. Furthermore, the DSS 1H signal may be used to indirectly reference 13C and 15N shifts using a simple ratio calculation [1]. Unfortunately, many biomolecular NMR spectroscopy labs use non-standard methods for determining the 1H, 13C or 15N “zero-point” chemical shift position. This lack of standardization makes it difficult to compare chemical shifts for the same protein between different laboratories. It also makes it difficult to use chemical shifts to properly identify or assign secondary structures or to improve their 3D structures via chemical shift refinement. Chemical shift re-referencing offers a means to correct these referencing errors and to standardize the reporting of protein chemical shifts across laboratories.
Probabilistic Approach for protein NMR Assignment Validation (PANAV) is a freely available stand-alone program that is used for protein chemical shift re-referencing. Chemical shift referencing is a problem in protein nuclear magnetic resonance as >20% of reported NMR chemical shift assignments appear to be improperly referenced. For certain nuclei these referencing issues can cause systematic chemical shift errors of between 1.0 and 2.5 ppm. Chemical shift errors of this magnitude often make it very difficult to compare NMR chemical shift assignments between proteins. It also makes it very hard to structurally interpret chemical shifts. Unlike most other chemical shift re-referencing tools PANAV employs a structure-independent protocol. That is, with PANAV there is no need to know the structure of the protein in advance of correcting any chemical shift referencing errors. This makes PANAV particularly useful for NMR studies involving novel or newly assigned proteins, where the structure has yet to be determined. Indeed, this scenario represents the vast majority of assignment cases in biomolecular NMR. PANAV uses residue-specific and secondary structure-specific chemical shift distributions that were calculated over short fragments of correctly referenced proteins to identify mis-assigned resonances. More specifically, PANAV compares the initial chemical shift assignments to the expected chemical shifts based on their local sequence and expected/predicted secondary structure. In this way, PANAV is able to identify and re-reference mis-referenced chemical shift assignments. PANAV can also identify potentially mis-assigned resonances as well. PANAV has been extensively tested and compared against a large number of existing re-referencing or mis-assignment detection programs. These assessments indicate that PANAV is equal to or superior to existing approaches.
PREDITOR is a freely available web-server for the prediction of protein torsion angles from chemical shifts. For many years it has been known that protein chemical shifts are sensitive to protein secondary structure, which in turn, is sensitive to backbone torsion angles. torsion angles are internal coordinates that can be used to describe the conformation of a polypeptide chain. They can also be used as constraints to help determine or refine protein structures via NMR spectroscopy. In proteins there are four major torsion angles of interest: phi, psi, omega and chi-1. Traditionally protein NMR spectroscopists have used vicinal J-coupling information and the Karplus relation to determine approximate backbone torsion angle constraints for phi and chi-1 angles. However, several studies in the early 1990s pointed out the strong relationship between 1H and 13C chemical shifts and torsion angles, especially with backbone phi and psi angles. Later a number of other papers pointed out additional chemical shift relationships with chi-1 and even omega angles. PREDITOR was designed to exploit these experimental observations and to help NMR spectroscopists easily predict protein torsion angles from chemical shift assignments. Specifically, PREDITOR accepts protein sequence and/or chemical shift data as input and generates torsion angle predictions for phi, psi, omega and chi-1 angles. The algorithm that PREDITOR uses combines sequence alignment, chemical shift alignment and a number of related chemical shift analysis techniques to predict torsion angles. PREDITOR is unusually fast and exhibits a very high level of accuracy. In a series of tests 88% of PREDITOR’s phi/psi predictions were within 30 degrees of the correct values, 84% of chi-1 predictions were correct and 99.97% of PREDITOR’s predicted omega angles were correct. PREDITOR also estimates the torsion angle errors so that its torsion angle constraints can be used with standard protein structure refinement software, such as CYANA, CNS, XPLOR and AMBER. PREDITOR also supports automated protein chemical shift re-referencing and the prediction of proline cis/trans states. PREDITOR is not the only torsion angle prediction software available. Several other computer programs including TALOS, TALOS+ and DANGLE have also been developed to predict backbone torsion angles from protein chemical shifts. These stand-alone programs exhibit similar prediction performance to PREDITOR but are substantially slower.
ShiftX is a freely available web server for rapidly calculating protein chemical shifts from protein X-ray coordinates. Protein chemical shift prediction is particularly useful in verifying protein chemical shift assignments, adjusting mis-referenced chemical shifts, refining NMR protein structures and assisting with the NMR assignment of unassigned proteins that have either had their structures determined by X-ray or NMR methods.
